Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
The plant toxins, ricin and abrin and the bacterial toxins, Shiga and Shiga-like toxins are type II ribosome inactivating proteins that inhibit protein synthesis by removing a highly conserved adenine from the a-sarcin/ricin loop (SRL) of the large rRNA. They consist of a catalytic A chain covalently joined by a disulfide bond to a cell binding B chain and are highly toxic to eukaryotic cells. Ricin naturally exists in the seeds of Ricinus communis (castor bean), a plant native to Asia, the Middle East and southern Europe.
In the castor bean, ricin A and B chains are encoded by a single gene, which is translated into a pre-proprotein of 576 amino acids. The ricin precursor consists of a 35 residue N-terminal extension, which contains the signal sequence. The mature RTA, which consists of 267 residues, is joined to the 262 residue mature RTB by a 12 residue linker peptide. The signal peptide directs the protein into the endoplasmic reticulum (ER) where pro-ricin is core glycosylated and disulfide bonds are formed within the protein. Four disulfide bonds form within the RTB sequence and the fifth one joins RTA with RTB in the ricin holotoxin.
The B-chain of ricin (RTB) is a lectin that binds galactose or N-acetylgalactosamine receptors on the surface of target cells and promotes subsequent endocytosis of the A-chain (RTA). After RTB binds to its receptor on the surface of animal cells, a portion of the endocytosed RTA reaches the Golgi complex. RTA is an N-glycosidase that depurinates ribosomes in the cytosol by removing a specific adenine (A4324 in rat 28S rRNA) from the highly conserved SRL in the large rRNA (7, 8). RTA undergoes retrograde transport from the Golgi to the endoplasmic reticulum (ER) and is thought to enter the cytosol from the ER.
The depurination of the SRL has been reported to interfere with the elongation factor 1 (eEF-1) dependent binding of amino acyl-tRNA to the ribosome, as well as the GTP-dependent binding of elongation factor 2 (eEF-2) and inhibit protein synthesis at the translocation step. There is evidence that ricin induces apoptosis in a wide variety of animal cells by mechanisms other than protein synthesis inhibition. Ricin-induced apoptosis in HeLa cells was associated with oxidative stress, glutathione depletion and activation of the caspase 3 cascade, followed by downstream events leading to apoptotic cell death.
Since ricin and many other AB-toxins are quite stable, one or a few molecules are sufficient to kill cells. RTA has been used in cancer therapy as the active moiety of immunotoxins selectively targeted to cancer cells. Due to its potent cytotoxicity and wide availability, ricin has been exploited as a biological weapon and an agent of bioterrorism and has been classified as a level B biothreat by the Centers for Disease Control and Prevention. Inhalation of small amounts of ricin aerosol can rapidly and irreversibly damage cells of the respiratory tract, leading to severe pulmonary incapacitation or death.